The glycine receptor (GIyR), is a member of the nicotinicoid receptor superfamily, which include the homologous nicotinic acetylcholine receptor, the 5-HT3 serotonin receptor, and the GABAA receptor, all of which act in rapid mediation of signal transduction at the synapse. This receptor is a glycine-gated anionic channel, and is the major inhibitory neurotransmitter channel in spinal cord and lower brain. The overall goal of this CEBRA proposal is the determination of human alpha1 GlyR structure at high resolution. These investigations of GlyR structure aim to provide structure-function information at a molecular level. Since this receptor family is targeted by anesthetics, alcohols, inhaled solvents and other narcotics, these structural determinations will impact on our understanding of the basic mechanisms underlying ion channel inhibition, desensitization, and activation. The CEBRA mechanism of funding is especially relevant since in the absence of diffractable microcrystals, funding is typically unavailable. Yet, dedicated higher-risk efforts such as those described in this proposal require support for successful high-resolution determination of membrane protein structure. The proposed studies exploit the previously developed expression system (Cascio et al., 1993). Successful overexpression and reconstitution of homomeric GlyR (Cascio et al., 2001) presents a unique opportunity to undertake structural studies of the GlyR. Preliminary studies noted a cholesterol-dependent conformational change in GlyR. Additionally, our coupled proteolysis and mass spectrometry studies (Leite et al., 2000) and spectroscopic studies of reconstituted GlyR (Cascio et al., 2001) have determined that the four-transmembrane helix model for the nicotinicoid receptors may be inappropriate. We propose to use recent advances in membrane protein crystallography exploiting lipidic cubic mesophases, co-crystallizations with monoclonal antibodies and/or crystallographic studies of a soluble form of the ligand-binding domain of the receptor. Given the difficulties in determining high-resolution structures of membrane proteins by crystallographic methods, we also alternatively propose to further refine receptor topology via determination of experimental constraints. These distance constraints will be determined using an EDTA-strychnine reagent or site-directed Cys mutagenesis coupled with chemical modification studies and mass spectrometry. Overall these investigations aim to provide insight into the general conserved structure of nicotinicoid channels.